Algae, a keystone of the aquatic food chain, have a rich and balanced content of many health promoting nutrients, including vitamins such as vitamin E and vitamin B, minerals such as iron and calcium, and carotenoids such as carotene and xanthophylls. In addition, they contain large amounts of essential amino acids, polysaccharides, and high quality lipids, especially very long-chain poly-unsaturated fatty acids and arachidonic acids.
As a result, algae have become increasingly useful for a variety of purposes. For example, algae biomass is an excellent source of animal feed, useful in livestock, larviculture, hatchery, and aquarium operations. Algae cells also comprise a variety of bio-chemicals, useful for the production of nutritional supplements, pharmaceuticals, and cosmetics. In addition, they serve as a promising source of clean and renewable energy, for example as raw materials for the production of biofuels (via pyrolysis of lipids). Algae biomass can be further used as inexpensive biomaterials for the passive removal of toxins, organic pollutants, and heavy metals from the water system. It has been estimated that the worldwide market size of algae products exceeds five billon dollars annually (Pulz and Gross 2004).
Bioprocess algae include those algae strains that are scaleable and commercially viable for production on a large scale. One well-known green unicellular bioprocess microalgae is Dunaliella. It is recognized for its commercial use in producing carotenoids such as beta-carotene and also glycerol for fine chemicals, foodstuff additives, and dietary supplements. Dunaliella is known to be composed of approximately 50% protein, 35% carbohydrate, and 8% lipids (A. Ben-Amotz, “Production of β-carotene and vitamins by the halotolerant alga Dunaliella,” Marine Biotechnology, Vol 1. Pharmaceutical and Bioactive Natural Products, D. H. Attaway and O. R. Zaborsky, eds., 1993; pg 413-414).
One Dunaliella strain particularly of interest is Dunaliella salina. The unicellular green alga Dunaliella salina is a member of the phylum Chlorophyta, class Chlorophyceae, order Dunaliellales, family Dunaliellaceae, with some 22 species of Dunaliella recognized (M. A. Borowitza and C. J. Siva. The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales) with emphasis on the marine and halophilic species. J. Appl. Phycol. 19:567-590; 2007). It has two flagella of equal length inserted anterior on the cell body, which is usually ovoid in shape but can vary with growth conditions. The cell lacks a rigid cell wall but is covered with a glycocalyx-type mucilage largely present on older cells. One large, cup-shaped posterior chloroplast with a pyrenoid is present in A stigma is laterally at the anterior part of the chloroplast. UTEX 1644 is considered a type strain of D. salina (M. A. Borowitza and C. J. Siva, supra.). The lipid content of the type-strain D. salina UTEX 1644 ranged from 3% to 6% on a dry-weight basis (A. Markovits, M. P. Gianelli, R. Conejeros, S. Erazo. Strain selection for beta-carotene production by Dunaliella. World J. Microbiol. Biotechnol. 9:534-537; 1993). The fatty acids are mostly C16 and C18 hydrocarbons, with a minor amount of longer-chain fatty acids.
The ability of Dunaliella to proliferate in high salt and high pH media at high temperatures allows scaleable, mass cultivation, notably in open ponds and raceways common to commercial production of other algae and cyanobacteria. In these conditions, the Dunaliella face little competition from predators or contaminating microalgae. The alga can be grown in seawater, brackish water, and also down to low salt conditions. Factors affecting cultivation are described in, for example, U.S. Pat. No. 4,115,949. Specific factors affecting production of Dunaliella parva for oil and for nitrogen-rich residue are taught in U.S. Pat. No. 4,341,038, for example, such that cultivation proceeds in 6% to 25% NaCl and in the presence of carbonic anhydrase enzyme derived from such algae.
One major obstacle in the commercialization of algae-derived compounds is the relative low productivity of the desired algae components and the high cost associated with the cultivation process. For example, conventional lipid-producing algae strains only contain about 3% to 6% of lipids on a dry weight basis. Further, there is a lack of effective cultivation methods capable of producing the desired algae component at a high yield without reducing total biomass production. For instance, conventional techniques utilize stress conditions to maximize the desired metabolite production, although the induction of stress simultaneously limits the biomass productivity. For example, productivity of Dunaliella total biomass cultured in paddle-wheel raceway ponds under stress conditions decreases to about 5 to 10 g DW per square-meter per day; whereas the biomass productivity is estimated to be 25 g DW per square-meter per day under non-stress conditions. For another example, under intense light and near-saturation salt concentrations, yield of Dunaliella beta-carotene can be significantly increased; however, under such conditions, the biomass yield decreases further to about 0.05 to 0.1 g DW per square-meter per day (A. Ben-Amotz, “Production of β-carotene and vitamins by the halotolerant alga Dunaliella,” Marine Biotechnology, Vol 1. Pharmaceutical and Bioactive Natural Products, D. H. Attaway and O. R. Zaborsky, eds., pg 413-414; 1993).
To address this problem, U.S. Pat. No. 4,958,460 employs a two-stage protocol: a first stage of non-stress cultivation under normal salinity to achieve maximal biomass production, and a second stage of stress cultivation under increased salinity. However, such two-stage protocols are less than ideal.
Another factor inhibiting the commercial production of bioprocess algae is the lack of live, certified, concentrated seedstock for bioprocess algae growers. As live algae concentrates are highly perishable, developing effective preservation means would significantly reduce the cost associated with the transportation and storage of algae cells. The art has utilized various techniques such as centrifugal concentrating, freezing, or freeze-drying of algae slurry for preservation. Use of various cryoproteactants such as DMSO and glycerol and preservatives such as methanol, ethanol, propanol, ethyl maltol, acetaldehyde, and glycerine has been attempted. Disadvantageously, algae pastes produced by these conventional preservation means are generally not viable. In addition, they need to be stored under stringent conditions, such as under refrigeration or freezing at a low temperature, thereby significantly increasing the cost of production.
In addition, separation of the cultivated algae from the culture medium is required for subsequent processing of the algal biomass. Many means for separation of the algae from the growth medium are known in the art, such as use of floating suction dredgers and thickening drums or filters. Harvesting of halophilic, unicellular, swimming microalgae by separating the majority of water from the algae-salt water slurry proceeds by centrifugation, filtration, or flocculation effected by increasing the pH of the algae-salt water slurry, as described, for example, in U.S. Pat. No. 4,341,038. The above techniques can be varied by employing variable NaCl concentrations and flotation, as described, for example, in U.S. Pat. Nos. 4,438,592 and 4,554,390. 6,936,459 teaches harvesting of algae by use of polyelectrolytes and forced flotation using compressed air. However, there remains need for additional harvesting methods.
In view of the above described state of the art, a substantial need exists for novel algae strains having high levels of desired bio-components and methods capable of producing algae-derived components with high yields and at a low cost. Further, novel means for the preservation and harvesting of live algae concentrates are needed. As will be clear from the disclosure that follows, these and other benefits are provided by the present invention.